The present invention describes a process for the preparation of autocrosslinked compounds of hyaluronic acid and the derivatives thereof by the technique of precipitation induced by supercritical antisolvent (SAS).
These crosslinked compounds of hyaluronic acid and the derivatives thereof can be used to advantage for the preparation of biomaterials for use in the field of medicine and surgery and in tissue engineering for medical and surgical purposes.
Hyaluronic acid is a heteropolysaccharide composed of alternating residues of D-glucuronic acid and N-acetyl-D-glycosamine. It is a straight-chained polymer with a molecular weight that varies between 50,000 and 13,000,000 Da according to, the source from which it is obtained and the methods of preparation and determination used. It is naturally present in the pericellular gels, in the fundamental substance of connective tissue and in vertebrate organisms, of which it is one of the chief components, in the synovial fluid of joints, in the vitreous humor, in the human umbilical cord tissues and in rooster combs.
Hyaluronic acid plays a vital role in many biological processes such as tissue hydration, proteoglycan organisation, cell differentiation, proliferation and angiogenesis (J. Aigner et al., L. Biomed. Mater. Res. 1998, 42, 172-181).
It is known that hyaluronic acid fractions can be used to facilitate tissue repair, as substitutes for the intraocular fluid, or they can be administered by the intra-articular route to treat joint pathologies, as described in European Patents Nos. 0138572 and 0535200.
Hyaluronic acid plays a fundamental role in the tissue repair process, especially in the early stages of granulation, stabilising the coagulation matrix and controlling its degradation, favouring the recruitment of inflammatory cells such as polymorphonucleate leukocytes and monocytes, of mesenchymal cells such as fibroblasts and endothelial cells and, lastly, orientating the successful migration of epithelial cells.
It is known that the application of hyaluronic acid solutions is able to accelerate healing in patients suffering from sores, wounds and burns. The role of hyaluronic acid in the various stages of the tissue repair process has been described by the construction of a theoretical model by Weigel, P. H. et al.: xe2x80x9cA model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healingxe2x80x9d, J. Theor: Biol:, 119, 219, 1986.
The use of low-molecular-weight hyaluronic acid fractions for the preparation of pharmaceutical compositions with bone-inducing properties (U.S. Pat. No. 5,646,129) is also known.
Hyaluronic acid derivatives maintain all the properties of the above glycosaminoglycan, with the advantage that they can be processed in various forms and that their solubility and degradation times can be varied according to the percentage of their derivation (EP 0216453 B1).
Moreover, also known are the total or partial esters of hyaluronic acid and the autocrosslinked derivatives of hyaluronic acid, their use in the pharmaceutical and cosmetic fields and in that of biodegradable materials (U.S. Pat. Nos 4,851,521; 4,965,353; 5,676,964).
The process known to date for the preparation of autocrosslinked derivatives of hyaluronic acid has the disadvantage that it is difficult to achieve homogeneous autocrosslinking throughout the mass of the product. Indeed, said process consists in preparing a solution of hyaluronic acid salt with tetrabutylammonium (HA-TBA) in an organic solvent such as N-methyl-pyrrolidone (NMP), dimethylsulfoxide (DMSO) or dimethylformamide (DMF) at a temperature of below 0xc2x0 C., agitating constantly while a solution of 2-chloro-1-methyl-pyridinium iodide (CMPJ) is added to whichever solvent or organic solvent is being used, the quantity of crosslinking agent being 25% that of the polymer mass. As soon as contact is made, the CMPJ produces crosslinking in the HA-TBA and, as its viscosity increases the solid becomes segregated before the crosslinking agent is homogeneously distributed. It therefore tends to settle preferentially on the solid and is no longer able to penetrate into the solution, leading to uneven crosslinking.
The ability of antisolvent fluids such as carbon dioxide (CO2) to solubilise in organic solvents under increased pressure has been exploited, in the present invention, to try to control the crosslinking reaction which is characterised by very rapid kinetics, especially in the early stage of mixing the reagents.
Rendering liquids far less viscous by swelling them makes it possible to achieve a mixture more rapidly and more efficiently than by the traditional process.
By compressing the antisolvent fluid it is possible to work in a swollen, and therefore dispersed environment and to control the crosslinking reaction once it has begun, slowing it down.
Addition of the crosslinking agent to the swollen mass is very rapid because of a specially designed injection system. Because of the swelling caused by the compressed antisolvent, the agitation of the mass and the fast injection of the crosslinking agent, it is possible to control the rate of crosslinking at the onset at temperatures of between 0 and 20xc2x0 C., preferably 10xc2x0 C.
At this temperature, the viscosity of the medium is such as will allow good homogenisation of the mass, unlike in the case of the conventional process that has to be conducted at temperatures of around xe2x88x9220xc2x0 C.
It is essential to work below precipitation pressure so that the separation of the polymer from the liquid phase occurs through the effect of the crosslinking and is not induced by precipitation due to the antisolvent effect; in the latter case, the crosslinking agent is only able to trigger the reaction superficially because it is unable to penetrate within the precipitate.
The present invention describes a process for the preparation of autocrosslinked compounds of hyaluronic acid and the derivatives thereof by means of precipitation using the supercritical antisolvent (SAS) technique.
These crosslinked compounds of hyaluronic acid and the derivatives thereof can be used to advantage for the preparation of biomaterials for use in the medical and surgical fields and in tissue engineering in the medical-surgical field.
The equipment used the technique is illustrated in FIG. 1, wherein P=precipitator; PI=CO2 pump; P2=pump for the crosslinking agent; VM1, VM2=millimetric valves for flow adjustment; VNR=cut-off valve; V1=on-off valve; VT1, VT2=three-way valves; A=sampling loop; PE=post-expansion vessel; R=rotameter.
The process according to the present invention involves the following steps:
a) preparing solutions of quaternary ammonium salt of hyaluronic acid or the derivatives thereof and the crosslinking agent;
b) loading the solutions of quaternary ammonium salt and crosslinking agent into the precipitator and into the container respectively, the latter being fitted with a pump to suck up the liquid;
c) adjusting the rotation speed to within a range of 200-1000 rpm, preferably between 250 and 450 rpm;
d) adjusting the temperature to within a range of xe2x88x9220xc2x0 C. to 20xc2x0 C., preferably between 0 and 10xc2x0 C.;
e) adjusting the pressure of the fluid to below that of precipitation of the quaternary ammonium salt of hyaluronic acid or the derivatives thereof, which depends upon the solvent and the temperature;
f) injecting the solution of crosslinking agent into the precipitator at a greater pressure than that present in the precipitator in order to disperse all the crosslinking agent homogeneously in the starting polymer solution;
g) optionally, injecting a buffer solution into the precipitator;
h) increasing the temperature to+ between 20 and 50xc2x0 C., preferably between 15 and 40xc2x0 C.;
i) leaving it to react for between two and twelve hours, preferably six hours;
j) washing the product with a flow of antisolvent at a pressure of between 60 and 150 bar, preferably between 80 and 100 bar;
k) depressurising and harvesting the product.
The following hyaluronic acid derivatives are to be preferred:
partial esters of hyaluronic acid wherein part of the carboxy functions are esterified with alcohols of the aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic series (EP 0216453 B1);
partial esters of hyaluronic acid wherein part of the carboxy functions are esterified with an arylaliphatic alcohol and the second part with long-chain, straight aliphatic alcohols with between 10 and 22 carbon atoms (WO 98/08876)
the partially O-sulfated (WO 95/25751) and/or N-sulfated derivatives (WO 98/45335);
the amide derivatives of hyaluronic acid.
The preferred quaternary ammonium salts are tetrabutylammonium salts. The crosslinking agents are, for example, 2-chloro-1-methyl-pyridinium iodide, 2-chloro-pyridine, 2-chloro-1-isopropyl-pyridinium iodide, 1-fluoro-2,4-dinitrobenzene.
The solutions are prepared in organic solvents such as N-methyl-pyrrolidone (NMP), dimethylsulfoxide (DMSO), dimethylformamide.